In recent years, significant advances have been made in micro-column separation techniques, such as capillary electrophoresis (CE). Injecting a precise and reproducible volume is fundamental to any separation technique. The volume reproducibility of injection is generally expected to be less than two percent, and is often required to be less than one percent to be analytically useful. This task is especially difficult in CE due to the extremely small volumes reproduced, as well as the frequent employment of narrow-bore capillary columns having inner diameters less than 100 .mu.m.
In capillary electrophoresis, injection of the samples usually requires that the inlet end of the separation capillary be removed from the electrolyte vial and installed in the sample vial. The liquid sample analyte is subsequently moved from the sample vial into the source end of the capillary, either by applying pressure to the sample vial and forcing the liquid into the end of the capillary or by applying a voltage differential between the sample solution, the destination vial, and across the capillary. This voltage differential electrokinetically drives the ionic analytes into the end of the capillary. The inlet end of the separation capillary is then removed from the sample vial and reinstalled in the inlet electrolyte vial.
In electrokinetic injection or electromigration, an end of the capillary and an electrode are placed into the sample and a voltage is briefly applied, causing a small band of sample to electromigrate into the capillary. This technique is generally reliable, and only depends on reproducible viscosity of the sample solution, reasonably constant ionic strength of the sample, and reproducible applied voltage. While all these requirements are reasonable to achieve, this technique of sample injection suffers from discrimination within the sample because solutes or analyte ions with higher mobilities will preferentially migrate into the electrophoresis column, and therefore change the relative composition of the sample.
Accordingly, pressure injection techniques or controlled pressure differentials over the separation column may be preferred in many instances. Generally, pressure injection has been induced either by gravity flow or siphoning, or by applying a constant vacuum or overpressure to the source or destination end of the connecting column.
In the gravity injection method, the source end of the capillary and the sample vial are positioned at controlled differential heights above the destination end of the capillary such that gravity forces the sample liquid into the capillary at a controlled rate.
While this technique is generally reproducible in many situations, this method is problematic when applied to extremely small volumes, such as those employed in capillary electrophoresis. In these instances, the surface forces between the sample fluids and the capillary walls and reservoir walls have a greater adverse affect on the relatively small injected volumes.
Moreover, in gravity injection, the inlet end of the capillary must be capable of being moved relative the outlet end to use gravity to force the liquid into the capillary. However, more recent CE instrument designs employ cassette assemblies to support and mount the capillary. While these cassette assemblies effectively isolate the capillary to improve the necessary temperature control, the inlet end must be fixed relative to the outlet end which essentially eliminates gravity injection as an optional mode.
In the overpressure or vacuum technique, a number of parameters must be reproducible with a relatively high tolerance to achieve the required injection accuracy and reproducibility. In addition to a reliable seal, and a reproducible volume sample-to-sample, the injection pressure applied to the sample vial must be reproducible to within at least 0.1 psi while the time that pressure is applied must be reproducible to within at least 0.1 seconds. Moreover, the viscosity of the liquid sample must be reproducible, sample-to-sample, as well. Typical of these patented pressure injection devices may be found in U.S. Pat. Nos. 5,207,886; 5,217,590 to Lauer et al.; and European Patent Application Publication No. 0,339,781 A2 to Burolla.
The Burolla system calculates the intended injection volume by measuring the pressure and time parameters during the injection, and then integrating the pressure-time curve. Hence, by determining the length of injection time required, at the applied real-time pressure, the intended volume injected into the capillary can be accurately estimated.
This system is advantageous in that the absolute pressure applied need not be accurately controlled, since the pressure is being measured real-time and since the time necessary to achieve the required injected volume can be varied, real-time, according to the actual pressure applied. Hence, during pressure fluctuations and undulations inherent in these systems, the length of time of the applied pressure can be varied to compensate for these fluctuations. This technique is capable of achieving reproducible injections with a Relative Standard Deviation (RSD) of less than two (2) percent.
One problem associated with these arrangements, however, is a potential lack of correlation between the actual volume injected and the estimated intended volume injected when very small injection volumes are concerned. The dynamic gain of the Beckman system is very high relative to very small injection volumes. In combination with the electronic and mechanical delays of the system, pressure fluctuations substantially decrease the volumetric accuracy for small volumes. Further, the relatively slow ramp-up times of pressure applied to the pressurized fluids and/or pressure fluctuations can cause variation in the dynamics of the sample injection process, which in turn adversely affect the accuracy and precision of the injected volumes.
In the Lauer system, the overpressure is applied to the inlet end of one of three capillary configurations to inject the liquid sample therein. U.S. Pat. No. 5,207,886 describes an injection device having a vacuum at the capillary outlet to perform the injection, while U.S. Pat. No. 5,217,590 discloses an injection system formed to apply overpressure in either a forward or rearward direction. In either system, a piston system is employed to generate the required pressure.
While this assembly is potentially very accurate, it is susceptible to system pressure leaks. This leakage is primarily caused by the repetitive sealing and unsealing at the sealed sample vials during handling which eventually deteriorates the seal integrity. Since the actual applied pressure is not measured or accounted for, even a small leak deleteriously affects the vial pressure causing variations in the actual injection volume, injection to injection.